REvIEwS - Advances in Clinical and Experimental Medicine

REvIEwS - Advances in Clinical and Experimental Medicine

Reviews
Adv Clin Exp Med 2012, 21, 3, 403–408
ISSN 1899–5276
© Copyright by Wroclaw Medical University
Grażyna Michalska-Krzanowska
Tryptase in Diagnosing Adverse Suspected
Anaphylactic Reaction*
Tryptaza w diagnostyce reakcji niepożądanych podejrzanych o anafilaksję
podczas znieczulenia oraz w okresie okołooperacyjnym
Division of Anesthesiology and Intensive Care, 111th Military Hospital with Outpatient Clinic Poznań, Poland
Abstract
Determination of serum mast cell tryptase (MCT) is becoming more widely used in diagnosing allergic reactions
involving mast cells. It can help evaluate the allergenic effects of drugs administered during anesthesia and the
perioperative period. Until now, data about the role of tryptase in the body has not been clarified yet. Patients with
elevated MCT levels should undergo further testing to find out the causative agent of a potential allergic reaction.
Patients with normal tryptase concentration should also undergo further diagnosis if they manifest clinical symptoms of a severe anaphylactic reaction (Adv Clin Exp Med 2012, 21, 3, 403–408).
Key words: tryptase, anaphylaxis, anesthesia, perioperative period.
Streszczenie
Oznaczenie stężenia tryptazy (Mast Cell Tryptase – MCT) w surowicy krwi znajduje coraz szersze zastosowanie
w diagnostyce reakcji alergicznych z udziałem komórki tucznej. Może być wykorzystywane w celu oceny alergizującego działania leków podawanych podczas znieczulenia oraz w okresie okołooperacyjnym. Dotychczas rola
tryptazy w organizmie nie została ostateczne określona. Pacjenci ze zwiększonym stężeniem MCT powinni być
poddawani dalszym badaniom w celu poszukiwania środka sprawczego, potencjalnej reakcji alergicznej. Pacjenci
z prawidłowym stężeniem tryptazy także powinni być poddawani dalszej diagnostyce, jeżeli mają kliniczne objawy
ciężkiej reakcji anafilaktycznej (Adv Clin Exp Med 2012, 21, 3, 403–408).
Słowa kluczowe: tryptaza, anafilaksja, znieczulenie, okres okołooperacyjny.
A huge increase in allergic diseases observed
in the last ten years significantly affects those with
allergic hypersensitivity to drugs administered
during the perioperative period and anesthesia.
According to data presented in the reference literature, the incidence of anesthesia-related anaphylaxis ranges from 1:3500 to 1:20000 anesthesias [1,
2]. Despite the lack of precise epidemiological reports from different regions, the growing risk of
anaphylactic reactions in the perioperative period
has been thoroughly observed. The importance of
the emerging problem of anesthesia-related anaphylaxis was particularly noticed in the Nordic
countries, Western Europe, Australia and New
Zealand [3, 4].
In 1960, the properties of mast cell granules
termed trypsin-like activity were discovered. The
isolated enzyme was named mast cell tryptase:
MCT [5]. Until now, data on the biological role of
tryptase in the body has not been definitively identified and clarified. At the same time, its function
in diseases involving mast cells has not yet been
fully explained. This results from the fact that the
physiological substrate which would react with
tryptase has not been discovered yet. Such an ability for some proteins has been demonstrated only
* This article is based on the Medline database from the years 1991–2010. When collecting literature, the author
used the following key words: tryptase, mast cell, anaphylaxis, mastocytosis.
404
G. Michalska-Krzanowska
under in vitro conditions. In the diagnosis of anaphylactic reactions, tryptase has been used as an
enzyme that monitors mast cell activity.
There are many chemicals involved in allergic
processes associated with the use of neuromuscular
blocking agents (NMBA) and other drugs administered in the perioperative period. Many different
drugs are used in rapid succession: not only anesthetics, but also antibiotics, fluids, nonsteroidal anti-inflammatory drugs and other compounds (e.g.
disinfectants, latex, chlorhexidine, etc.). Most of
these drugs are given intravenously and in bolus,
bypassing the body’s primary immune filters and
presenting high concentrations of antigen directly
to the mast cells and basophils. So it is difficult to
say which drug caused the suspected anaphylactic
reaction or if the reaction was the result of the additive side effects of several drugs injected simultaneously [1]. Tryptase, however, is perceived to be
of the highest importance. According to Scandinavian authors, determining the presence of this enzyme is a key element in diagnosing anaphylactic
reactions [4, 5]. The NMBA-related anaphylaxis
confirmed by the presence of tryptase in plasma
is compatible with a diagnosis resulting from skin
and serological tests.
Mast Cell
A mast cell (mastocyte, MC) is derived from
multipotential bone marrow cells CD34+. It appears in the peripheral blood, and then is deposited in tissues such as skin, lungs, intestinal mucosa
and submucosa. There it matures under the influence of general and local growth factors, such as
the stem cell factor (SCT), mast cell growth factor
(MCGF) and c-kit ligand. The population of mast
cells is not fully homogeneous. Two types of cells
are distinguished among them. The first type includes MCT cells (mast cells containing tryptase)
whose granules contain only tryptase (located in
pulmonary alveoli and intestinal mucosa). The
second type consists of MCTC cells (mast cells
containing chymase and tryptase) whose granules,
apart from tryptase, contain chymase (located in
the dermis, intestinal submucosal layer and blood
vessels) [6]. The numerical ratio of MCTC to MCT
is varied. It depends on the type of tissue. The ratio
is different under health and disease conditions.
MCTC predominate in the skin, while MCT prevail in lungs and gastrointestinal mucosa. Allergic
reactions involve MCT which migrate to the nasal
and bronchial epithelium [6].
A mastocyte is typically identified with an immediate hypersensitivity reaction, i.e. a type I reaction according to the Coombs and Gell classi-
fication, where it plays a key role [7]. Tryptase is
one of many mediators released from mast cells
involved in immediate-type reactions. [8, 9]. Other
substances stored in granules (preformed [including tryptase]), exhibit a pharmacological effect on
blood vessels of the respiratory tract and the respiratory tract itself, resulting in clinical symptoms of
hay fever, bronchial asthma, urticaria, or anaphylaxis.
Data presented in the literature also suggests
that mast cells are involved in inflammatory processes in the course of other diseases. Attention is
drawn to the role of mast cells in autoimmune diseases such as: rheumatoid arthritis, scleroderma or
pemphigoid.
The process of releasing substances stored
in granules is referred to as “degranulation”. Degranulation of mast cells is an active process of
secretion whose essential elements are energy and
calcium. A mastocyte can be activated both immunologically and non-immunologically. The former
can occur in two ways: in the mechanism dependent and independent of IgE antibodies. Mast cells
have membrane receptors for these antibodies.
When a mutually complementary antibody joins
the membrane receptor and then the mastocyte
contacts an allergen, the cell is degranulated [10,
11]. Many non-specific stimuli may, however, release its mediators from the cell. Among them are
trauma, high temperature and toxins.
The cytoplasm of a mastocyte contains numerous basophil granules which consist of such substances as histamine, serotonin, glycosaminoglycans, peroxidase, superoxide dismutase, eosinophil
chemotactic factors (ECF), neutrophil chemotactic
factors (NCF), tryptase, chymase, heparin and numerous cytokines.
The role of cytokines is associated with the initiation of the late allergic response (LAR), or the
chronic phase of allergic inflammation (chemotaxis, infiltration and accumulation of inflammatory cells). Clinically, the late allergic response
occurs analogically to the early symptoms. It can
begin 2–4 hours after the immediate allergic reaction. It peaks after about 8–12 hours and lasts up
to 24 hours.
Tryptase
Tryptase is an enzyme specific to mast cell
granules. The enzyme is present in complexes with
heparin and has a high molecular weight compared
to histamine (approximately 140 kDa). The genes
responsible for tryptase development and synthesis are located on the short arm of chromosome
16. In the human body, depending on the ami-
405
Tryptase in Anaphylactic Reactions
Table 1. Types of human tryptase [12, 13]
Tabela 1. Typy ludzkiej tryptazy [12, 13]
Type (Typ)
Subtype (Podtyp)
Characteristics (Charakterystyka)
α-tryptase
αI
α II
– exhibits little biological activity,
– present in low concentrations in blood serum even in the absence of mast
cell degranulation
β-tryptase
β1
β2
β3
– the main form stored in granules of mast cells,
– released during severe inflammation, such as anaphylactic shock. Absent in
serum
γ-tryptase
γI
γ II
a protein embedded in the cell membrane of both mast cell granules and its
outer membrane, its function is unknown
δ-tryptase
δI
ä II
the type has a significantly shorter chain and different affinity for peptides
no acid composition, it is present as: α-tryptase,
β-tryptase, γ-tryptase and δ-tryptase. Each of them
has subtypes. Table 1 presents the characteristics
of each type.
Resistance to protease inhibitors characteristic
of tryptase results from an unprecedented ability
of this protein to create molecules with a tetramer
structure and the active center facing the inside
and unreachable to proteins with inhibiting properties [14]. The regulation of tryptase activity due
to the specific protection mechanism is not subject
to protease-protease inhibitor dependence. The
only suggested regulatory mechanism, based on
observations in vitro, is the pH-dependent ability
of tryptase to bind to the heparin molecule, which
facilitates the formation of active tetrameres. At
neutral pH, this ability disappears, and tetramers
disintegrate. Acidic pH is conducive to forming
tetramers [15].
Studies describe the multidirectional effects
of the biological activity of tryptase. It causes the
disintegration of bronchodilator mediators, which
may result in a smooth muscle spasm in the respiratory track. In the endothelium of blood vessels, their nitric-oxide-dependent vasodilation
and increased permeability with the characteristics of hypotension have been observed. Tryptase
degrades the vasoactive intestinal peptide (VIP)
and calcitonin gene-related peptide (CGRP). It
also exhibits mitogenic activity for fibroblasts and
increases expression of the first vascular cellular
adhesion molecule (VCAM-1). Tryptase shows
activity against plasma coagulation factors (fibrinogen degradation, prekallikrein activation, kininogen distribution, activation of the pro-urokinasetype plasminogen activator (pro-uPA)), whereas
the end result is the anticoagulant activity [16].
Tryptase secretion from mast cells depends on
the availability of intra- and extracellular calcium
and magnesium resources, as well as the presence
of phospholipase A2 (PLA2) and phospholipase
C (PLC) inhibitors [17, 18].
Determination of tryptase concentration is
used as a marker of mast cell activation in inflammatory diseases as the enzyme can be described as
typical of mast cells. Available in the pharmacological market, the reference set (UniCAP 100 Phadia,
Pharmacia, Uppsala, Sweden) makes it possible to
determine the total tryptase concentration. It is
a fluorescence immunoassay [19]. Antitryptase reacts with tryptase in the patient serum specimen.
After washing, enzyme-labeled antibodies against
β-tryptase are added to form a complex. After incubation, unbound enzyme-antitryptase is washed
away and the bound complex is incubated with
a developing agent. At the end of the reaction the
fluorescence is measured, and the degree of fluorescence correlates with the amount of tryptase
present.
An increase in serum tryptase levels is observed between 3–6 hours after the onset of an
anaphylactic reaction. It returns to normal within 12–14 hours [20]. The practical significance
of this is the window of opportunity for testing
for systemic anaphylaxis. Tryptase testing can be
performed on blood samples obtained 1–6 hours
after onset of the reaction. The working party of
the Association of Anaesthetists of Great Britain
and Ireland on ‘suspected anaphylactic reactions
associated with anaesthesia’ advises taking three
samples [21]. One immediately after the initial resuscitation, another 1 hour after the start of the
reaction at the moment the MCT concentration
normally peaks and the last simple 24 hours after
the reaction to get a baseline value. The sample
taken 1 hour after the start of the reaction is obviously the most important.
406
G. Michalska-Krzanowska
The Application of Tryptase
Level Determination
The longer half-life of tryptase in serum makes
it a more convenient diagnostic marker than histamine. Histamine can also be released by other
cells such as basophils, antigen-presenting cells
and bacteria. The optimum time for tryptase determination is between 1–6 hours after the exposure to an allergen (peak secretion between the
fifteenth minute and the second hour, with a halflife of 1.5–2.5 hours). It should be remembered,
however, that a higher level of tryptase may be
observed in diseases and conditions other than
anaphylactic allergy. The examples can be wound
healing, scar formation involving the mast cell,
multiorgan trauma, hypoxia, myocardial infarction, heroin intoxication, mastocytosis. An increase in tryptase levels beyond systemic mastocytosis is also diagnosed in acute myeloid leukemia,
myelodysplastic syndromes, hypereosinophilic
syndrome related to FIP1L1-PDGFRA mutation,
the exogenous administration of SCF (stem cell
factor) and end-stage renal insufficiency with increased endogenous SCF levels. Systemic mastocytosis is characterized by mast cell hyperplasia in
bone marrow, skin, liver, spleen and gastrointestinal mucosa [22]. A study of tryptase levels in patients with biopsy-proven mastocytosis reported
concentrations of total tryptase > 20 ng.mL–¹ and
rations of total tryptase to β-tryptase > 20, whereas
normal patients had total tryptase levels < 14 ng.
mL–¹ [23]. The sensitivity of the total mast cell
tryptase concentration as a diagnostic test is 83%,
with a specificity of > 98%. Total serum tryptase
concentrations may be useful in evaluating treatment aimed to reduce mast cell numbers in patients with systemic mastocytosis.
Myelodysplastic syndrome (MDS) is a large
group of acquired neoplastic disorders of the bone
marrow most common in the elderly and is caused
by an abnormal differentiation and maturation of
haemopoetic cells. Serum tryptase levels can be
used to differentiate the MDS variants. Follow-up
studies should clarify whether an elevated serum
tryptase concentration in MDS is of prognostic
significance.
Serum tryptase determination can be used to
evaluate the allergenic activity of drugs taken during anesthesia and the perioperative period. A single tryptase level test can be insufficient, therefore,
in order to improve the specificity and sensitivity
of determination, a serial test and its comparison
with the basic concentration is suggested. It was
shown that suxamethonium did not cause the release of this substance from both the basophils of
peripheral blood and mast cells of the skin and
lungs. On the contrary, after a dose of atracurium
and vecuronium – the effect of tryptase release
was directly proportional to the dose used, and the
dynamics of concentrations stimulated by aminosteroid and benzylisoquinoline drugs was similar.
High intersubject variability was observed in atracurium activity in response to mast cell tryptase
secretion from the lungs. Among the analyzed
drugs, only atracurium resulted in the release of
histamine from mast cells obtained from the atria
of the heart. Intradermal stimulation of mast cells
with aminosteroid and benzylisoquinoline relaxants caused a dose-dependent biochemical effect
(tryptase release) and increase in objective (erythema) and subjective (pain, itching) symptoms. In
the case of suxamethonium and benzylisoquinolines, the occurrence and intensity of intradermal
stimulation subjective symptoms (itching, erythema and pain) corresponded with increasing concentrations of tryptase, which was not observed in
relation to aminosteroids [18, 24].
Tryptase was also used in the diagnosis of fatal
anaphylactic reactions. It is suggested that an elevated level of tryptase collected post-mortem may
persist up to the third day after death from anaphylaxis [25]. Such a determination of serum tryptase
in the event of a suspected anaphylactic reaction
is useful in most cases of parenteral penetration of
allergens. This was proved in post-mortem examinations carried out up to 24 hours after the death
of 19 victims. A tryptase level above 10 ng/ml was
found in nine out of nine victims of anaphylaxis
who were bitten by insects, two of two cases of
anaphylaxis after drugs given parenterally, and six
of eight cases of food allergy. At the same time,
a level below 5 ng.ml–¹ was observed in 57 cases of
post-mortem serum analysis in persons who died
of other reasons than anaphylaxis [25].
Determination of serum tryptase levels was
widely used in the diagnosis of mastocytosis.
A tryptase level above 25 ng.mL–¹ is one of the diagnostic criteria for the disease, although it is not
a specific marker of mastocytosis. The diagnosis of
this disease is also affected by clinical features and
a histopathological picture of the bone marrow.
A number of laboratory methods for determining tryptase levels in bodily fluids have been developed. The radioimmunoenzymatic method is the
most useful and the most common method which
makes it possible to detect β II-tryptase, specific to
allergic processes, at concentrations of less than
1 ng.mL–¹, and the range of determinability on the
calibration graph is 1–200 ng.mL–¹ [11]. It has been
shown that in the time interval up to 6 hours after
the first symptoms of anaphylaxis, there is no correlation between the time of blood collection and
Tryptase in Anaphylactic Reactions
the recorded tryptase levels [11, 19]. Kinetic parameters and enzyme stability under different external environmental conditions make it possible
to collect biological material, and then to conduct
quantification of tryptase not only at the time of
the onset of allergic symptoms. It is recommended
to collect blood for clot in the amount of 5–10 ml,
within 1–4 hours from the onset of allergic symptoms [4, 18, 19, 26]. It is recommended to compare
the resultant level of tryptase with a control value,
obtained at least 24 hours after an anaphylactic
reaction. Its elevated level in comparison with the
control value is a highly sensitive indicator of anaphylactic reaction in the perioperative period [4].
However, there is a group of patients with
clinical symptoms of severe anaphylactic reaction,
but their tryptase is maintained at normal levels.
They require further diagnostic tests. Namely, an
elevated tryptase level does not always correlate
with severe anaphylaxis. A negative test result of
a tryptase level does not justify a withdrawal from
further examination. No increase in tryptase concentration in each case of anaphylaxis is attributed
to its local release (e.g. in laryngeal edema) which
is not sufficient to increase the total serum concentration, or a greater participation of basophils than
mast cells in the pathomechanism of anaphylaxis
in a given patient.
Perioperative anaphylactic reactions are a very
important anesthesiological problem, because most
often they result from the effects of drugs used for
general anesthesia. The greatest risk of allergic reactions is associated with the anesthesia induction
phase, and skeletal muscle relaxants are one of the
allergenic factors. Aminosteroids trigger allergic
reactions more often than benzylisoquinolines –
constituting about 50% of cases, among them rocuronium (30%), vecuronium (17%) and pancuronium (6%). Among benzylisoquinolines, allergic
reactions most often occur after atracurium and
407
mivacurium, 20% and 6% respectively. The fewest
allergic reactions are observed after cisatracuium –
0.3% of cases. Depolarizing relaxants (suxamethonium) cause anaphylaxis as often as drugs from the
group of non-depolarizing aminosteroids.
In a case of suspected anaphylaxis during anesthesia, it is important to conduct three basic diagnostic tests: biochemical determination, serological
determination and skin tests. Tryptase concentration determination is a reliable and effective method of confirming the incidence of anaphylaxis.
The multidirectional effects of drugs used in
the perioperative period makes anaphylaxis a very
complex problem. This is a very topical issue, and
epidemiological data proves the problem is really
serious. The severity of an allergic reaction in the
final assessment of the suitability of measuring
tryptase level as an important element in the diagnosis of anaphylaxis in the perioperative period
should undoubtedly be taken into account. However, determination of tryptase levels can not be
the only diagnostic tool in perioperative anaphylaxis. A detailed medical history to define the causative agent and clarify the accompanying clinical
symptoms should play the primary role. The diagnosis of anaphylaxis is particularly difficult or even
impossible in the perioperative period due to the
lack of cooperation with the patient (influenced by
soporific drugs) and because of the large number of
medicines used both for anesthesia and perioperative care. The diagnosis of anaphylaxis in the perioperative period, despite the constant expansion
of knowledge, is still a difficult challenge in daily
clinical practice. Difficulties in assessing individual
risk of anaphylaxis result from a lack of sensitive,
widely available diagnostic methods confirming
an anaphylaxis episode, and the inability to distinguish between people who are allergic to an allergen and those in whom the exposure to a given
factor may cause a life-threatening reaction.
References
  [1] Hepner DL, Castells M: Anaphylaxis during the perioperative period. Anest Analg 2003, 97, 1381–1395.
  [2] Mertes PM, Laxenaire MC: Anaphylaxis during general anaesthesia – prevention and management. CNS Drugs
2000, 14, 115–133.
  [3] Guttormsen AB: Allergic reactions during anaesthesia – increased attention to the problem in Denmark and Norway. Acta Anaesthesiol Scand 2001, 45, 1189–1190.
  [4] Kroigaard M, Garvey LH, Gillberg L, Johansson SG: Scandinavian Clinical Practice Guidelines on the diagnosis,
management and follow-up of anaphylaxis during anaesthesia. Acta Anaesthesiol Scand 2007, 51, 655–670.
  [5] Harboe T, Guttormsen AB, Dybendal T: Anaphylaxis during anesthesia in Norway. Anesthesiology 2005, 102,
897–903.
  [6] Krishnaswamy G, Kelley J, Johanson D: The human mast cell: functions in physiology and disease. Frontiers
Biosci 2001, 6, 1109–1127.
  [7] Gell PG, Coombs RA, Lachmann PF: Clinical aspects of immunology. In: Atopy. Ed.: Pepys J. Oxford, Blackwell
Scientific. Ed 3 1975, 877–902.
  [8] Metcalfe DD, Baram D, Mekori YA: Mastcells. Physiol Rev 1997, 77, 1033–1079.
  [9] Galli SJ, Nakae S, Tsai M: Mast cell in the development of adaptive immune responses. Nat Immunol 2005, 6,
135–142.
408
G. Michalska-Krzanowska
[10] He S, Xie H: Modulation of tryptase release from human tonsil mast cells by protease inhibitors. Pharmacol Rep
2005, 57, 523–530.
[11] Payne V, Kam PCA: Mast cell tryptase: a review of its physiology and clinical significance. Anaesthesia 2004, 59,
695–703.
[12] Marone G: Human basophils and mast cells. Biological aspects. Dvorak AM ultrastructural analysis of human
mast cells and basophils. Chem Immunol 1995, 61, 1–28.
[13] Marone G: Human basophils and mast cells: Biological Aspects. MacGlashan DW – Signal transduction and cytokine production by human basophils. Chem Immunol Basel Karger 1995, 61, 88–113.
[14] Pereira PJ, Bergner A, Macedo-Ribeiro S, Hubner R: Human beta-tryptase is a ring-like tetramer with active
sites facing a central pore. Nature 1998, 392, 306–311.
[15] Hallgren J, Spillmann D, Pejler G: Structural requirements and mechanism for heparin-induced activation of
a recombinant mouse mast cell tryptase, mouse mast cell protease-6: formation of active tryptase monomers in
the presence of low molecular weight heparin. J Biol Chem 2001, 276, 42774–42781.
[16] He SH, Xie H, He YS: Induction of tryptase and histamine release from human colon mast cells by IgE dependent
or independent mechanisms. World J Gastroenterol 2004, 10, 319–322.
[17] Veien M, Szlam F, Holden J, Levy J: Mechanism of nonimmunological histamine and tryptase release from human cutaneous mast cells. Anesthesiology 2000, 92, 1074–1081.
[18] Stellato C, Paulis A, Cirillo R: Heterogeneity of human mast cells and basophils in response to muscle relaxants.
Anaesthesiology 1991, 74, 1078–1086.
[19] Enrique E, Garcia-Ortega P, Sotorra O: Usefulness of UniCAP tryptase fluoroimmunoassay in the diagnosis of
anaphylaxis. Allergy 1999, 54, 602–606.
[20] Schwartz LB, Yunginger JW, Miller J: Time course of appearance and disappearance of human mast cell tryptase
in the circulation after anaphylaxis. J Clin Invest 1989, 83, 1551–1555.
[21] The Association of Anaesthetists of Great Britain and Ireland: Suspected anaphylactic reactions associated with
anaesthesia, August 2003.
[22] Lange M, Renke J, Gleń J: Tryptasa mastocytowa, interleukina 6 jako wykładnik ciężkości przebiegu mastocytozy.
Post Dermatol Alergoz 2010, 27, 4, 238–245.
[23] Austin FK: Allergies, Anaphylaxis and systemic mastocytosis. In: Harrisons Principles of International Medicine.
Eds.: Braunwald E, Fauce AS, Kasper DL, 15th ed. New York: McGraw-Hill 2001, 1919–1920.
[24] Koppert W, Blunk J, Petersen L: Different patterns of mast cell activation by muscle relaxants in human skin.
Anaesthesiology 2001, 95, 659–667.
[25] Yunginger JW, Nelson DR: Laboratory investigation of deaths due to anaphylaxis. J Forensic Sci 1991, 36, 857–
865.
[26] Hallgren J, Pejler G: Biology of mast cell tryptase. FEBS J 2006, 273, 1871–1895.
Address for correspondence:
Grażyna Michalska-Krzanowska
Stroma 4c
70-004 Szczecin
Poland
E-mail: [email protected]
Conflict of interest: None declared
Received: 16.09.2011
Revised: 2.01.2012
Accepted: 6.06.2012